It is widely known that many chemotherapeutic regimens fail because the side-effects of the drugs used limit the dose that can be administered. This is particularly true of solid tumors. The clinically tolerated doses are often insufficient to kill all of the cells, thereby enriching the tumor population for drug resistant mutants. Among the surviving tumor cells in below-effective treatment regimens are mutant cells that arise spontaneously within the tumor cell population, and are resistant to the treatment drug. Each subsequent round of chemotherapy enriches the population for the resistant cells, which grow and continue to mutate, some to even higher levels of resistance. There is an established linear-log relationship between dose and tumor kill. The higher the dose of the drug, the greater the chance of eradicating the tumor. While methods have been developed to selectively target and kill tumor cells, many of the targeting methods either reduce the effectiveness of the drug, or call for a complex series of reactions to prepare a drug.
In the consideration of solid tumors, it should be recognized that local effective dosage, and systemic dosage, need not be the same. Thus, the only effective portion of the chemotherapeutic agent administered is that which reaches the tumor cell. Many chemotherapeutic agents are administered systemically, however, and only a limited portion (the local dosage) of the dosage administered actually reaches the cell. Thus, dose limitations frequently result in only a fraction of the permitted dosage actually reaching the cell.
The mechanism of inherent and acquired resistance of tumors to many forms of treatment involves glutathione. Elevated glutathione levels in tumors have been shown to contribute to resistance to chemotherapy and radiotherapy and prevent the initiation of the apoptotic cascade in tumor cells (1-5). The enzyme γ-glutamyl transpeptidase (GGT, EC 2.3.2.2), which is localized to the cell surface, cleaves the γ-glutamyl bond of extracellular glutathione, releasing glutamic acid and cysteinyl-glycine, thus enabling the cell to use extracellular glutathione as a source of cysteine for increased synthesis of intracellular glutathione (6). GGT is induced in many human tumors, enhancing their resistance to chemotherapy (7; 8) Inhibiting GGT prior to chemotherapy or radiation would sensitize GGT-positive tumors to treatment Inhibiting GGT for as little as 2 hours lowers the intracellular cysteine concentration in GGT-positive tumors (3). However, all known GGT inhibitors, prior to the present invention, are too toxic for use in humans at concentrations needed to inhibit GGT activity (9; 10).
GGT plays an essential role in releasing cysteine from extracellular glutathione. Most cells are unable to take up intact glutathione (6). In GGT knockout mice, the absence of GGT in the renal proximal tubules results in the excretion of glutathione in the urine (11). In these mice, the glutathione in the glomerular filtrate cannot be cleaved into its constituent amino acids for reabsorption. GGT knockout mice have a 4500-fold elevation of glutathione in their urine relative to their GGT-wild-type littermates. GGT knockout mice grow slowly and die by ten weeks of age due to a cysteine deficiency. GGT also metabolizes S-nitroso-glutathione, initiating the release of nitric oxide from S-nitroso-glutathione. Nitric oxide is known to play a role in asthma, vascular diseases and other pathologies. GGT contributes to inflammatory disease by converting leukotriene LTC4 to LTD4. In arthritis GGT stimulates osteoclast formation thereby increasing bone destruction in the joint. Further, GGT is involved in the degradation of glutathione during storage of blood and platelets.
GGT catalyzes the cleavage of γ-glutamyl compounds and the transfer of the gamma-glutamyl group to an acceptor substrate by a ping-pong kinetic mechanism (12). Glutathione and glutathione-S-conjugates are the most common physiologic substrates of GGT. They serve as the gamma-glutamyl donor in the initial reaction. In the first reaction the γ-glutamyl bond of the initial substrate is cleaved, the γ-glutamyl group becomes covalently bound to the enzyme and the remainder of the substrate is released as the first product. With glutathione as the substrate, cysteinyl-glycine is released and is subsequently cleaved into cysteine and glycine by cell surface dipeptidases. In the second reaction of GGT transpeptidation, the γ-glutamyl-group is transferred from the γ-glutamyl-GGT complex to the second substrate (the acceptor). Dipeptides and amino acids have the highest Km as acceptors. The second substrate with the covalently bound gamma-glutamyl group is released as the second product from the enzyme.
Compounds which are known to inhibit GGT include the glutamine analogues acivicin, 6-diazo-5-oxo-L-norleucine, and azaserine (13). Rational design of GGT inhibitors based on studies of the active site has led to the identification of additional γ-glutamyl analogues. Lherbet and Keillor have designed sulfur derivatives of L-glutamic acid which inhibit GGT (14; 15). Han and coworkers have synthesized and tested a series of γ-(monophenyl)phosphono glutamate analogues which also functioned as inhibitors of GGT (16; 17).
Evaluation of several of the glutamine analogues that inhibit GGT has shown them to be toxic (9; 10). Acivicin, the most potent inhibitor of GGT that has been tested clinically, is a neurotoxin (18). The neurotoxicity of the glutamine analogues may be due to interference with glutamine in recycling the neurotransmitter glutamate via the glutamate-glutamine cycle. Glutamine is also involved in the synthesis of several nucleotides and complex polysaccharides. Inhibition of these essential synthetic pathways can be toxic to dividing cells. Acivicin, 6-diazo-5-oxo-L-norleucine, and azaserine all cause bone marrow suppression (9). There is no previously known GGT inhibitor that can be used clinically. Identification of GGT inhibitors which could be used clinically has been a highly desired, yet unmet, need, until the present invention.
Physicians generally prescribe three main treatments for cancer: surgery, radiation therapy, chemotherapy or a combination of these.
Surgery is generally advisable when physicians can safely remove the cancer from the body. In situations where the cancerous cells have spread, surgeons sometimes must remove large areas of healthy tissue along with the tumor to insure that no malignancy remains. In these cases, physicians may remove lymph nodes from the tumor area because cancer can spread through nodes. However, unfortunately many cancers are discovered too late for surgical cure. In many cases, the patient does not experience symptoms until the cancer has progressed to a malignant stage.
Radiation therapy is used to destroy cancer cells. However, radiation can both cause and destroy cancer and can cause damage to surrounding tissues. Side effects of radiation therapy include radiation sickness, which are nausea and skin redness in the tumor area. Reducing the negative side effects of radiation treatment is therefore highly desirable.
Chemotherapy uses drugs that take advantage of cancer cells' rapid growth and consumption of large amounts of nutrients. Chemotherapy side effects include nausea and temporary full or partial hair loss. Antimetabolites, one group of these drugs, work by mimicking the nutrients the body's cells consume. Physicians inject these drugs into the bloodstream, where they travel throughout the body, consumed by every cell. Rapidly growing cancerous cells consume much more of the poisonous drugs than do normal cells. As a result, the drugs destroy cancerous cells faster than normal cells. Another group of chemotherapy drugs interferes with the duplication of DNA (cells reproduce by duplicating their genetic code, or DNA), so cells cannot reproduce. Chemotherapy can also be directed against mutated proteins in the tumor cells, overexpressed proteins or other properties of the tumor cell. Chemotherapy drugs act on all the patients cells, the cancerous cells and the healthy cells. A physician's challenge is to administer the drugs to kill only the cancer cells, not the healthy cells. Side effects such as those described above prevent the long term or recurrent use of these drugs. Furthermore, there are an increasing number of effective drugs that can no longer be used due to resistance by the causative agent. It is thus highly desirable to reduce the side effects of therapeutics while maintaining the cancer-reducing qualities thereof, enabling the longer term use, or use of higher dosages of the drugs.